For the analyses of proteins, one of the present inventors reported a method of using a compound represented by the following formula:
wherein R1, R2 and R3 are the same or different and each is hydrogen, halogen or alkyl, or a salt thereof as a labeling compound (patent document 1). Specifically, three compounds represented by the following formulas:
which have mass difference from each other due to isotope labeling (which are generically referred to as Py compound) have been synthesized. In the method described in the document, a protein in two or more analysis samples are labeled with any of the Py compounds to confer a mass difference to the same protein among samples, the samples are mixed, and the protein is hydrolyzed. The obtained labeled peptide is subjected to mass spectrometry to identify the protein from which it derives by structural analysis of the peptide, and the presence ratio of the identified protein among samples is further calculated based on the mass spectrum.
On the other hand, conventional analysis of amino acid employs a quantification method including mutual separation of 20 or more kinds of biological amino acids by one performance of high performance liquid chromatography (HPLC) analysis, reacting the amino acids with ninhydrin, and quantifying the amino acids based on the absorbances of the resultant products. However, the sensitivity of this method is low, and is not suitable for the analysis of an amino acid at a low concentration.
To achieve higher sensitivity, therefore, use of a fluorescent label has been proposed, which realizes about 10-fold or higher sensitivity.
Furthermore, for an amino acid present at a low concentration undetectable even thereby, a method including separation using liquid chromatography followed by mass spectrometry is adopted. In this case, to facilitate ionization of separated amino acids, amino acids are derivatized using a reagent that reacts with an amino group, and then subjected to liquid chromatographic separation and mass spectrometry. However, even when this method is used, the conventional technique has a limitation in highly sensitive analysis.
Patent document 2 describes a method of producing various pyrylium compounds. It is stated that the formula described in the document produces two kinds of isomers; however, since separation of the both is not easy, the detection relies on the NMR analysis of a pyridine compound produced by a reaction with ammonia and that an acid usable for the reaction has Ho of −10 to −5.